Enhanced fibrinolysis detection in a natural occurring canine model with intracavitary effusions: Comparison and degree of agreement between thromboelastometry and FDPs, D-dimer and fibrinogen concentrations.

Dogs with intracavitary effusion have coagulative abnormalities indicative of primary fibrinolysis/hyperfibrinolysis. The aim of this case control study was to investigate by rotational thromboelastometry (ROTEM) and standard coagulation tests (fibrin-fibrinogen degradation products, D-dimer and fibrinogen) fibrinolysis in dogs with intracavitary effusions. Thirty-two dogs with intracavitary effusion and 32 control sick dogs without effusion were studied. Frequency of fibrinolysis grade of severity (i.e., hypofibrinolysis/basal fibrinolysis vs increased fibrinolysis vs hyperfibrinolysis) by ROTEM and standard coagulation tests were compared between groups. Pattern of fibrinolysis by ROTEM (i.e., late vs intermediate vs fulminant) and type of fibrinolysis by standard coagulation tests (i.e., hypofibrinolysis/basal fibrinolysis vs primary fibrinolysis vs secondary fibrinolysis vs primary hyperfibrinolysis vs secondary hyperfibrinolysis) were also compared between groups. Dogs with intracavitary effusion had a lesser degree of hypofibrinolysis and basal fibrinolysis and a higher degree of increased fibrinolysis and hyperfibrinolysis compared to controls, both by ROTEM and by standard coagulation tests (P = 0.042 and P = 0.017, respectively). Nevertheless, there was a poor agreement between the two classification schemes (34.4%, K = 0.06, 95% CI = -0.14 ‒ +0.26). Dogs with intracavitary effusion showed, by ROTEM, a lesser degree of hypofibrinolysis and basal fibrinolysis and a higher degree of late, intermediate, and fulminant fibrinolysis compared to controls (P = 0.044). Finally, dogs with intracavitary effusion had, by standard coagulation tests, a higher frequency of primary fibrinolysis and primary hyperfibrinolysis and a lower frequency of secondary fibrinolysis compared to controls. Dogs with intracavitary effusion showed an increased frequency and a different and more severe pattern of fibrinolysis compared to controls.

Introduction

Coagulation and fibrinolysis are precisely regulated by the measured participation of substrates, activators, inhibitors, cofactors and receptors. Fibrinolysis is the process whereby stable fibrin strands are broken down by plasmin [1]. Basal fibrinolysis, the ongoing removal of fibrin deposits, ensures blood fluidity while preventing blood loss. Physiologic fibrinolysis is localized fibrinolysis in response to thrombosis, and is necessary for the re-establishment of blood flow [2]. Physiologic fibrinolysis is mediated by fibrin-bound plasmin [2]. Primary fibrinolysis develops independently of intravascular activation of coagulation, and plasmin is formed without concomitant formation of thrombin [3]. It is mediated by plasma-free plasmin. Generalized fibrinogenolysis occurs when the production of plasmin within the general circulation overwhelms the neutralizing capacity of antiplasmins, potentially leading to severe bleeding, a disorder known as primary hyperfibrinolysis [2], primary hyperfibrinogenolysis [2,4,5], or pathologic fibrinolysis [2]. Finally, when fibrinolysis occurs as an appropriate response to persistent thrombin generation it is termed secondary or reactive fibrinolysis. This phenomenon keeps blood vessels patent by resolving redundant clots [6], and it is present in virtually every patient with disseminated intravascular coagulation [2]. Secondary fibrinolysis has also been reported in inflammatory diseases, such as sepsis [2,7,8].

In the clinical setting, having elevated plasma fibrin-fibrinogen degradation products (FDPs) with a normal D-dimer concentration has been suggested as a possible indicator of primary fibrinolysis/hyperfibrinolysis [4,9,10]. We recently demonstrated that dogs with ascites or pleural effusion had abnormalities of their fibrinolytic system indicative of primary fibrinolysis/hyperfibrinolysis, based on coagulation tests [11– 13]. In fact, intracavitary effusions, which have a demonstrated inherently fibrinolytic activity in humans, dogs, and horses [11-21] are continuously exchanged with the systemic circulation [14,22]. Therefore, upon re-entering into the circulatory system the intracavitary fluids might contribute to the enhanced fibrinolysis detected in these dogs, as previously documented to be the case in humans [14,15,23-27].

Fibrinolytic activity can also be assessed by viscoelastic hemostasis analyzers such as rotational thromboelastometry (ROTEM). Rotational thromboelastometry is a point-of-care device that rapidly detects systemic changes in in vivo coagulation. In the ROTEM, coagulation is activated with ellagic acid (INTEM test) or tissue factor (EXTEM test). This is carried out to standardize the in vitro coagulation process and subsequent fibrinolysis. The latter can be quantified by clot lysis parameters such as the EXTEM lysis index at 60 minutes (LI60) and the EXTEM maximum lysis (ML) [28]. LI60 is the percentage residual clot firmness at 60 minutes after the thromboelastometric coagulation time, which is the time in seconds from the test start until a clot firmness of 2 mm is obtained. ML is the percentage reduction in maximum amplitude of clot firmness reached during the run time [28]. In addition, EXTEM clot amplitude at 5 minutes (A5), clot amplitude firmness 5 minutes after a clot firmness amplitude of 2 mm has been reached, can be used for early detection of fibrinolysis [28], and identifying patients developing hyperfibrinolysis [29].

The aims of this study were to assess the following hypotheses. First, we hypothesized that ROTEM could detect an enhanced and more severe pattern of fibrinolysis in dogs with intracavitary effusions compared to dogs without effusion. Second, we hypothesized that there was an agreement in fibrinolysis severity detection between the combination of ROTEM assay results and fibrinogen concentrations, or alternatively by concentrations of FDPs, D-dimers and fibrinogen. Finally, we also hypothesized that dogs with intracavitary effusions had more primary fibrinolysis/hyperfibrinolysis compared to dogs without effusion when assessed by concentrations of FDPs, D-dimers and fibrinogen.

Material and methods

Animals

In this case control study, systemic fibrinolytic activity in dogs was assessed by ROTEM and standard plasma coagulation tests. A comparison was made between dogs with ascites and/or pleural effusions and control sick dogs without any type of intracavitary effusion. Group 1 included dogs with any type of abdominal and/or pleural effusion, which was confirmed by thoracic radiography, ultrasonography or computer tomography, and who presented to the San Marco Veterinary Clinic from March 2008 to November 2016. Intracavitary effusions in group 1 dogs were classified according to their pathophysiology of formation. Diagnosis of the disease causing the intracavitary effusion was used as the criterion to establish the pathophysiology of the fluid formation. Group 2 included sick dogs without any type of intracavitary effusion. They were chosen from the electronic medical database, P.O.A System-Plus 9.0®, and included dogs that presented to the San Marco Veterinary Clinic during the same period of time as those in group 1. All control dogs were individually matched to group 1 dogs type of underlying disease based on their. When 2 or more dogs fulfilled these criteria, the choice for the pairing was randomly made by the computer system P.O.A System-Plus 9.0®. To be included in the study, dogs of both groups were required to have received a specific diagnosis relevant to the initial reason for examination. They also had a complete medical record, including history and results of physical examination. Assays completed at the time of presentation were a complete blood count (including blood smear examination), serum biochemistry analysis, standard coagulation tests and ROTEM assay, and urinalysis. Dogs of both groups were excluded from the study if they had been treated with plasma, plasma derivates, anticoagulant therapy, tranexamic acid, or for anticoagulant intoxication within the 30 days prior to study enrolment.

Localization and type of effusion in dogs of groups 1, and causes of sickness for both groups of dogs were described. Gender, sexual status, age, pure breed vs. mongrel status, body weight and frequency of clinically relevant bleeding for all the dogs included in the 2 groups were recorded and used in comparisons. For the purpose of this study, clinically relevant bleeding was defined as any bleeding that caused anemia, defined as a hematocrit (HCT) < 39.0% (reference interval 39.0–59.2).

Analysis of ROTEM and standard plasma coagulation tests

Venous blood samples for ROTEM point of care and plasma standard coagulation tests were taken from the cephalic (for medium/large-size dogs) or jugular (for small size-dogs) veins. In all dogs, venous blood samples for both ROTEM and standard plasma coagulation tests were taken at the same time (± 2 hours). Two 3.5 mL aliquots of blood were transferred in plastic tubes, containing 3.2% sodium citrate (3.2% sodium citrate Vacuette® 3.5 mL, Grenier Bio-One, Kremsmünster, Austria) to give a final ratio of volumes of anticoagulant to blood of 1:9, for the measurement of both ROTEM and standard plasma coagulation tests. ROTEM parameters analyzed in this study included A5, LI60 and ML for a 2-hour runtime (ML/2h) in EXTEM assays. Plasma standard coagulation tests analyzed in this study included fibrinogen, semiquantitative FDPs, and D-dimer concentrations.

EXTEM assays on ROTEM (Pentapharm GmbH, Munich, Germany) were performed according to the manufacturer’s recommendations. Analyses were started within 10 minutes of blood sampling. Briefly, according to the pipetting programme, 20 μL re-calcification reagent (0.2 mmol/L calcium chloride solution; StarTEM, TEM Innovations GmbH, Munich, Germany) and 20 μL EXTEM activation reagent (human recombinant tissue factor; EXTEM, TEM Innovations GmbH, Munich, Germany) were added to a pre-warmed cup. Then, 300 μL whole blood containing 0.32% sodium citrate was added to the cup and, after a semi-automated mixing step, the cup holder was placed in the measuring position of the ROTEM device. The measurements, run at 38.0°C, were stopped after 120 min according to the protocol. After measurement, all ROTEM tracings were visually evaluated for artefacts. Of the parameters measured and calculated by the ROTEM device, EXTEM A5, LI60 and ML/2h were further evaluated by statistical analysis.

Tubes with whole blood containing 0.32% sodium citrate were centrifuged at 1,950 g for 5 min, plasma was harvested, and standard coagulation tests analysis was performed within 1 hour of blood sample collection. Plasma fibrinogen concentrations were determined by quantitative assays, using STA Fibrinogen (Diagnostica Stago, Asnières sur Seine, France), with a STA-R Evolution automated analyzer (Diagnostica Stago, Roche, Bäsel, Switzerland). The detection limit for fibrinogen was 60 mg/dL; for statistical analysis values below this concentration were entered into the data sheet as 59 mg/dL. Plasma concentrations of FDPs were determined using a semiquantitative plasma latex agglutination kit, FDPs Plasma, (Diagnostica Stago, Asnières sur Seine, France) that was validated for use with canine blood [30,31]. Plasma D-dimer concentrations were determined using a validated [32], immunoturbidimetric quantitative assay, Tina-quant D-dimer (Roche Diagnostic GmbH, Mannheim, Germany), with an Olympus AU 2700 automated analyzer (Olympus Diagnostics, Hamburg, Germany).

For all ROTEM and standard plasma coagulation tests, internal laboratory reference intervals were calculated from 40 and 120 clinically healthy randomly selected dogs, respectively.

All collection procedures were performed solely as part of the dog’s prescribed health care and for standard diagnostic and monitoring purposes. Previous informed written consent was obtained from all dog owners. No anesthesia, euthanasia, or any kind of animal sacrifice was required in this study. All procedures complied with the European Union legislation “on the protection of animals used for scientific purposes” (Directive 2010/63/EU) and with the ethical requirements of Italian law (Decreto Legislativo 04/03/2014, n. 26). Accordingly, this study did not require authorization or an ID protocol number.

Fibrinolysis definitions

Based on the ROTEM assay results and plasma fibrinogen concentration, fibrinolysis severity grading for this study was:

Hypofibrinolysis and basal fibrinolysis were LI60 values higher or within the internal laboratory reference interval of 86% - 98%, respectively.

Increased fibrinolysis was LI60 values smaller than the internal laboratory reference interval of 86% with plasma fibrinogen concentration > 100 mg/dL.

Hyperfibrinolysis was LI60 smaller than the internal laboratory reference interval of 86% with plasma fibrinogen concentration ≤ 100 mg/dL.

From the categorization introduced by Schöchl et al., for dogs with LI60 values smaller than the internal laboratory reference interval of 86%, 3 patterns of fibrinolysis were identified, based on the time course of clot breakdown: (a) fulminant with a total breakdown of the clot within 30 minutes, (b) intermediate with a total breakdown between 30 and 60 minutes, and (c) late with a breakdown of the clot after 60 minutes [33].

Based plasma semiquantitative FDPs, D-dimer, and plasma fibrinogen concentrations, fibrinolysis severity grading for this study was:

Hypofibrinolysis or basal fibrinolysis had normal FDPs and D-dimer concentrations.

Increased fibrinolysis had increased FDPs and/or D-dimer concentrations with fibrinogen concentration > 100 mg/dL. Increased fibrinolysis had further sub-types:

Primary fibrinolysis had increased FDPs and normal D-dimer concentrations with fibrinogen concentrations > 100 mg/dL;

Secondary fibrinolysis had normal to increased FDPs and increased D-dimer concentrations with fibrinogen concentrations > 100 mg/dL.

Hyperfibrinolysis had increased FDPs and/or D-dimer concentrations with fibrinogen concentration ≤ 100 mg/dL. Hyperfibrinolysis had further sub-types:

Primary hyperfibrinolysis had increased FDPs and normal D-dimer concentrations with fibrinogen concentrations ≤ 100 mg/dL;

Secondary hyperfibrinolysis had normal to increased FDPs and increased D-dimer concentrations with fibrinogen concentrations ≤ 100 mg/dL.

Statistical analysis

Continuous data were assessed for normality of distribution with the Shapiro-Wilk test. Normally distributed data are reported as a mean ± standard deviation (SD), and non-normally distributed data were reported as a median and interquartile range (IQR).

Differences of sexual status (χ2-test), pure breed vs mongrel dogs (Fisher exact test), age (t-test), body weight (t-test), and clinically relevant bleeding (χ2-test with Yates correction) were evaluated between groups 1 and group 2.

EXTEM A5 (t-test), LI60 (Mann-Whitney test), ML/2h (Mann-Whitney test), plasma fibrinogen (Mann-Whitney test), semiquantitative FDPs (χ2-test), and D-dimer (Mann-Whitney test) concentrations were compared between group 1 and group 2.

Differences between group 1 and 2 in fibrinolysis grade of severity (hypofibrinolysis or basal fibrinolysis, increased fibrinolysis and hyperfibrinolysis) assessed by ROTEM assay results and plasma fibrinogen concentration and by plasma semiquantitative FDPs, D-dimer, and fibrinogen concentrations were evaluated by Fisher exact test. Agreement in fibrinolysis grade of severity assessed by these 2 methods was evaluated by Cohen’s Kappa statistic.

Differences between group 1 and 2 in pattern of fibrinolysis (hypofibrinolysis or basal fibrinolysis, late fibrinolysis, intermediate fibrinolysis and fulminant fibrinolysis) assessed by ROTEM assay results were evaluated by Fisher exact tests.

Differences between group 1 and group 2 in type of fibrinolysis (hypofibrinolysis or basal fibrinolysis, primary fibrinolysis, secondary fibrinolysis, primary hyperfibrinolysis and secondary hyperfibrinolysis) assessed by plasma semiquantitative FDPs, D-dimer, and fibrinogen concentrations were evaluated by Fisher exact test.

Clinically relevant bleeding can cause hypoperfusion and the latter has been associated with hyperfibrinolysis in both humans and dogs [34,35]. Therefore, viscoelastic measures of fibrinolysis (i.e., EXTEM A5, LI60, and ML/2h), standard plasma coagulation tests (i.e., plasma fibrinogen, semiquantitative FDPs, and D-dimer concentrations), ROTEM fibrinolysis grade of severity, and ROTEM pattern of fibrinolysis were also evaluated excluding dogs with clinically relevant bleeding from both groups. Group 1 and 2 without clinically relevant bleeding dogs were named group 1A and 2A, respectively.

For all statistical analyses, the significance level was set to α = 0.05.

Results

During the study period, 1619 dogs with any type of abdominal and/or pleural effusion, as confirmed by thoracic radiography, ultrasonography or computer tomography, presented to the clinic. For 34 of these dogs, plasma samples for both standard coagulation tests and ROTEM assay were collected at presentation. Two dogs were excluded from further analysis, 1 due to rodenticide exposure and 1 due to tranexamic acid treatment. The remaining 32 dogs entered the study in group 1. Twenty-five of these dogs had only abdominal effusions, including 2 transudates due to decreased colloid osmotic pressure, 10 transudates due to increased hydrostatic pressure, 3 exudates, and 10 hemorrhagic effusions. Six dogs had concomitant abdominal and pleural effusions, including 5 transudates due to increased hydrostatic pressure and 1 hemorrhagic effusion in both the abdominal and the thoracic cavities. One dog had only a pleural effusion, an exudate. Dogs with intracavitary effusions (group 1) and sick dogs without any type of intracavitary effusion (group 2) were matched for type of underlying disease which included in each group: neoplasia (n = 15), cardiac problems (5), liver disease (3), inflammatory or immune-mediated disease (3), traumatic disease (3), gastrointestinal disorders (2), and sepsis and infectious disease (1).

Group 1 dogs included 17 males, 16 (50%) sexually intact and 1 (3.1%) neutered, and 15 females, 3 (9.4%) sexually intact and 12 (37.5%) spayed. Twenty (62.5%) dogs were pure breed and 12 (37.5%) were mongrels. Mean age was 107 ± 40 months and mean body weight was 26.7 ± 15.1 kg. Clinically relevant signs of bleeding were present in 13 of these dogs. One case was trauma-related, 11 secondary to rupture of pathologic/neoplastic organs, 1 secondary to rupture of pathologic/neoplastic organs and concurrent severe thrombocytopenia, and in 1 case secondary to idiopathic pericarditis.

Group 2 dogs included 14 males, 7 (21.9%) sexually intact and 7 (21.9%) neutered, and 18 females, 5 (15%) sexually intact and 13 (40%) spayed. Seventeen (53.1%) dogs were pure breed and 15 (46.9%) were mongrels. Mean age was 108 ± 47 months and mean body weight was 22.1 ± 14.7 kg. Clinically relevant signs of bleeding were present in 4 of these dogs. One case was secondary to trauma, 1 secondary to rupture of pathologic/neoplastic organs, 1 secondary to severe thrombocytopenia, and 1 secondary to surgery in a dog affected by von Willebrand disease.

There was a statistical difference regarding sexual status (χ2 = 8.56, P = 0.036) with more intact males in group 1 compared to group 2. There was an overall 53% breed match between groups 1 and group 2 with no statistical difference found in the percentage of pure breed vs mongrel dogs (P = 0.61), age (t = -0.07, P = 0.94), and body weight (t = 1.23, P = 0.22). Frequency of clinically relevant bleeding was significantly higher in dogs with intracavitary effusions compared to the controls (χ2 = 5.13, P = 0.023).

ROTEM assay and plasma fibrinogen, FDPs and D-dimer concentrations

EXTEM A5 (reference interval, 29–44 mm) was significantly smaller in group 1 (mean = 34.88 ± 16.23 mm) compared to group 2 (mean = 53.41 ± 16.24 mm; t = -4.57, P < 0.001; Fig 1A). EXTEM LI60 (reference interval 86%– 98%) was significantly lower in group 1 (median = 95%, IQR 61% ‒ 100%) compared to group 2 (median = 99%, IQR 97% ‒ 99%; U = 366, P = 0.047; Fig 1B). EXTEM ML/2h (reference interval 19%– 38%) was significantly higher in group 1 (median = 18.5%, IQR 5% ‒ 47%) compared to group 2 (median = 6%, IQR 3% ‒ 14%; U = 290, P = 0.003; Fig 1C). Finally, also when dogs with clinically relevant bleeding were excluded from analysis EXTEM A5, LI60, and ML/2h, remained significantly smaller, lower and higher, respectively, in group 1A compared to group 2A (Table 1).

Fig 1

Tukey boxplots of 3.2% citrated whole blood EXTEM A5 (A), 3.2% citrated whole blood EXTEM LI60 (B), 3.2% citrated whole blood EXTEM ML/2h (C), plasma fibrinogen concentrations (D), and plasma D-dimer concentrations (E) from dogs with intracavitary effusion (n = 32) and control sick dogs without intracavitary effusion (n = 32). A: Data distributions of the two groups of dogs are significantly different (P < 0.001). B: Data distributions of the two groups of dogs are significantly different (P = 0.047). C: Data distributions of the two groups of dogs are significantly different (P = 0.003). D: Data distributions of the two groups of dogs are significantly different (P < 0.001). E: Data distributions of the two groups of dogs are not significantly different (P = 0.262). The shaded regions represent the reference interval for each test. The bottom and top of the box are the 1st and 3rd quartiles; the median is the black line inside the box. Whiskers correspond to the lowest datum still within 1.5 IQR of the lower quartile, and the highest datum still within 1.5 IQR range of the upper quartile. Black dots are outlier values (> 1.5 IQR away from the closest end of the box). IQR: interquartile range.

Table 1

ROTEM and standard plasma coagulation tests evaluated excluding dogs with clinically relevant bleeding.

Parameters	Group 1A	Group 1B	Test values	P-values
	(n = 19)	(n = 28)
EXTEM A5 (mm)
mean ± SD	40.47 ± 15.9	53.82 ± 16.47	t = -2.76	P = 0.008
(RI = 29–44)
EXTEM LI60 (%)
median (IQR)	87 (32 ‒ 99)	99 (97 ‒ 96)	U = 402	P = 0.003
(RI = 86–98)
EXTEM ML/2h (%)
median (IQR)	27 (9 ‒ 80)	7 (3 ‒ 15)	U = 96	P < 0.001
(RI = 19–38)
Fibrinogen (mg/dL)
median (IQR)	187 (95 ‒ 248)	373 (267 ‒ 574)	U = 92	P < 0.001
(RI = 152–284)
Semiquantitative	<5, n = 7	<5, n = 14
FDPs (μg/mL)	5–20, n = 8	5–20, n = 8	χ2 = 1.04	P = 0.592
(RI < 5)	> 20, n = 4	> 20, n = 6
D-dimer (μg/mL)
median (IQR)	0.8 (0.01 ‒ 0.12)	0.15 (0.01 ‒ 0.43)	U = 209	P = 0.222
(RI = 0.01–0.34)

Group 1A: dogs with intracavitary effusion without clinically relevant bleeding; group 2A: sick dogs without intracavitary effusions and without clinically relevant bleeding; IQR: interquartile range; RI: reference interval.

Plasma fibrinogen concentrations (reference interval, 152–284 mg/dL) were significantly lower in group 1 (median = 177 mg/dL, IQR 96 ‒ 234 mg/dL) compared to group 2 (median = 371 mg/dL, IQR 276 ‒ 569 mg/dL; U = 144.5, P < 0.001; Fig 1D). No statistical difference was found in plasma concentrations of semiquantitative FDPs (reference interval < 5 μg/mL) between group 1 and group 2 (χ2 = 3.23, P = 0.199; Table 2). No statistical difference was also found in plasma D-dimer concentrations (reference interval 0.01–0.34 μg/mL) between group 1 (median = 0.09 μg/mL, IQR 0.02 ‒ 0.2 μg/mL) and group 2 (median = 0.16 μg/mL, IQR 0.03 ‒ 0.54 μg/mL; U = 429, P = 0.262; Fig 1E). Finally, also when dogs with clinically relevant bleeding were excluded from analysis plasma fibrinogen concentrations remained significantly lower in group 1A compared to group 2A, while no statistical difference was found in plasma concentrations of semiquantitative FDPs and D-dimer concentrations (Table 1).

Table 2

Plasma concentrations of semi quantitative FDPs in dogs with intracavitary effusions and control sick dogs without intracavitary effusions.

	FDPs (μg/mL)
	(RI <5)
	< 5	≥ 5 < 20	≥ 20
Group 1	9 (28.12%)	11 (34.38%)	12 (37.50%)
(n = 32)
Group 2	16 (50.0%)	8 (25.0%)	8 (25.0%)
(n = 32)

Data are the No. (%) of dogs. Group 1: dogs with intracavitary effusions; group 2: sick dogs without intracavitary effusions. RI: reference interval.

Fibrinolysis

There was a significant difference in fibrinolysis grade of severity (i.e., hypofibrinolysis or basal fibrinolysis, increased fibrinolysis, and hyperfibrinolysis) assessed by ROTEM assay results and plasma fibrinogen concentrations between group 1 and group 2 (P = 0.042), with group 1 dogs having a lesser degree of hypofibrinolysis and basal fibrinolysis and a higher degree of increased fibrinolysis and hyperfibrinolysis compared to dogs of group 2 (Table 3). This difference was still present when dogs with clinically relevant bleeding were excluded from analysis, with dogs of group 1A having a lesser degree of hypofibrinolysis and basal fibrinolysis and a higher degree of increased fibrinolysis and hyperfibrinolysis compared to dogs of group 2A (P = 0.012; Table 3). ROTEM pattern of fibrinolysis (i.e., hypofibrinolysis or basal fibrinolysis, late fibrinolysis, intermediate fibrinolysis and fulminant fibrinolysis) was significantly different between group 1 and group 2 (P = 0.044), with group 1 dogs having a lesser degree of hypofibrinolysis and basal fibrinolysis and a higher degree of late, intermediate, and fulminant fibrinolysis compared to dogs of group 2 (Table 4). This difference was still present when dogs with clinically relevant bleeding were excluded from analysis, with dogs of group 1A having a lesser degree of hypofibrinolysis and basal fibrinolysis and a higher degree of late, intermediate, and fulminant fibrinolysis compared to dogs of group 2A (P = 0.009; Table 4).

Table 3

Differences in fibrinolysis grade assessed by ROTEM in dogs with intracavitary effusions and control sick dogs without intracavitary effusions.

	Hypofibrinolysis	Basal fibrinolysis	Increased fibrinolysis	Hyperfibrinolysis
Group 1	12 (37.5%)	9 (28.1%)	5 (15.6%)	6 (18.8%)
(n = 32)
Group 2	17 (53.1%)	12 (37.5%)	2 (6.3%)	1 (3.1%)
(n = 32)
Group 1A	5 (26.3%)	5 (26.3%)	4 (21.1%)	5 (26.3%)
(n = 19)
Group 2A	15 (53.6%)	10 (35.7%)	2 (7.1%)	1 (3.6%)
(n = 28)

NB: for statistical analysis dogs with hypofibrinolysis and basal fibrinolysis were grouped together. Data are the No. (%) of dogs. Group 1: dogs with intracavitary effusions; group 2: sick dogs without intracavitary effusions; group 1A: dogs with intracavitary effusion and without clinically relevant bleeding; group 2A: sick dogs without intracavitary effusions and without clinically relevant bleeding.

Table 4

Differences in pattern of fibrinolysis assessed by ROTEM in dogs with intracavitary effusions and control sick dogs without intracavitary effusions.

	Hypofibrinolysis	Basal fibrinolysis	Increased or hyperfibrinolysis
			Late	Intermediate	Fulminant
Group 1	12 (37.5%)	9 (28.1%)	6 (18.8%)	4 (12.5%)	1 (3.1%)
(n = 32)
Group 2	17 (53.1%)	12 (37.5%)	3 (9.4%)	0 (0%)	0 (0%)
(n = 32)
Group 1A	5 (26.3%)	5 (26.3%)	5 (26.3%)	3 (15.8%)	1 (5.3%)
(n = 19)
Group 2A	15 (53.6%)	10 (35.7%)	3 (10.7)	0 (0%)	0 (0%)
(n = 28)

NB: for statistical analysis dogs with hypofibrinolysis and basal fibrinolysis were grouped together. Data are the No. (%) of dogs. Group 1: dogs with intracavitary effusions; group 2: sick dogs without intracavitary effusions; group 1A: dogs with intracavitary effusion and without clinically relevant bleeding; group 2A: sick dogs without intracavitary effusions and without clinically relevant bleeding.

There was also a significant difference in fibrinolysis grade of severity between group 1 and group 2 (P = 0.017) when this was assessed by plasma semiquantitative FDPs, D-dimer, and fibrinogen concentrations, with group 1 dogs having a lesser degree of hypofibrinolysis and basal fibrinolysis and a higher degree of hyperfibrinolysis compared to dogs of group 2 (Table 5). This difference was still present when dogs with clinically relevant bleeding were excluded from analysis, with dogs of group 1A having a lesser degree of hypofibrinolysis and basal fibrinolysis and a higher degree of hyperfibrinolysis compared to dogs of group 2A (P = 0.016; Table 5).

Table 5

Differences in fibrinolysis grade of severity and type assessed by plasma semi quantitative FDPs, D-dimer, and fibrinogen concentrations in dogs with intracavitary effusions and control sick dogs without intracavitary effusions.

	Hypofibrinolysis/ basal fibrinolysis	Increased fibrinolysis		Hyperfibrinolysis
		PF	SF	PHF	SHF
Group 1	8 (25.0%)	15 (46.9%)	2 (6.25%)	5 (15.6%)	2 (6.25%)
(n = 32)
Group 2	12 (37.5%)	10 (31.25%)	10 (31.25%)	0 (0%)	0 (0%)
(n = 32)
Group 1A	6 (31.6%)	8 (42.1%)	0 (0%)	4 (21.0%)	1 (5.3%)
(n = 19)
Group 2A	11 (39.3%)	9 (32.1%)	8 (28.6%)	0 (0%)	0(0%)
(n = 28)

NB: for statistical analysis dogs with PF and SF and dogs with PHF and SHF were grouped together only to assess differences in fibrinolysis grade of severity between groups 1 and 2 and between groups 1A and 2A. To assess type of fibrinolysis no subgroups were assembled together. Data are the No. (%) of dogs. Group 1: dogs with intracavitary effusions; group 2: sick dogs without intracavitary effusions; group 1A: dogs with intracavitary effusion and without clinically relevant bleeding; group 2A: sick dogs without intracavitary effusions and without clinically relevant bleeding. PHF: primary hyperfibrinolysis; PF: primary fibrinolysis, SHF secondary hyperfibrinolysis; SF: secondary fibrinolysis.

Within the 64 dogs included in the study and grouped together, the observed concordance in fibrinolysis grade of severity, assessed by ROTEM assay results and fibrinogen concentrations and by plasma semiquantitative FDPs, D-dimer, and fibrinogen concentrations, was 34.4%. There was a poor agreement between the 2 classification schemes (K = 0.06, 95% CI = -0.14 ‒ +0.26; Table 6).

Table 6

Observed concordance in fibrinolysis grade of severity assessed by ROTEM assay results and fibrinogen concentrations and by plasma semi quantitative FDPs, D-dimer results and plasma fibrinogen concentrations in the 64 dogs included in the study.

Fibrinolysis grade of severity assessed by		ROTEM assay results and fibrinogen concentration
		Hypofibrinolysis / basal fibrinolysis	Increased fibrinolysis	Hyperfibrinolysis
Plasma semi quantitative FDPs, D-dimer and fibrinogen concentrations	Hypofibrinolysis / basal fibrinolysis	14	3	3
	Increased fibrinolysis	33	4	0
	Hyperfibrinolysis	3	0	4

There was a significant difference in type of fibrinolysis (i.e., hypofibrinolysis or basal fibrinolysis, primary fibrinolysis, secondary fibrinolysis, primary hyperfibrinolysis and secondary hyperfibrinolysis), assessed by plasma semiquantitative FDPs, D-dimer, and fibrinogen concentrations, between group 1 and group 2 (P = 0.004), with group 1 dogs having a higher frequency of primary fibrinolysis and primary hyperfibrinolysis and a lower frequency of secondary fibrinolysis compared to dogs of group 2 (Table 5). This difference was still present when dogs with clinically relevant bleeding were excluded from analysis, with dogs of group 1A still having a higher frequency of primary fibrinolysis and primary hyperfibrinolysis and a lower frequency of secondary fibrinolysis compared to dogs of group 2A (P = 0.004; Table 5).

Discussion

The primary aim of this study was to assess if ROTEM could detect enhanced fibrinolytic activity in dogs with intracavitary effusions. The EXTEM A5, LI60, and ML/2h results supported the presence of an enhanced systemic fibrinolytic state in these dogs, as already detected by the use of the combination of plasma FDPs, D-dimers and fibrinogen concentrations in our previous studies [11-13]. The enhanced systemic fibrinolytic activity in dogs with intracavitary effusion was still preset also when animals with clinically relevant bleeding were excluded from analysis from both groups, suggesting that this result was not exclusively driven by a possible hypoperfusion due to hemorrhage. EXTEM A5 correlates with platelet count and fibrinogen concentration [36], and can be used for early detection of fibrinolysis [28]. An EXTEM A5 ≤ 35 mm can identify more than 90% of human patients developing hyperfibrinolysis [29]. Our findings of a smaller EXTEM A5 and a lower fibrinogen concentration in dogs with intracavitary effusion would therefore be in agreement with these reports. Moreover, the lower EXTEM LI60 and the higher EXTEM ML/2h found in these dogs would suggest that the smaller EXTEM A5 and the lower fibrinogen concentration were, at least in part, due to an enhanced fibrinolytic state, also making EXTEM A5 an early marker of fibrinolysis in dogs.

Schöchl et al. [33], and other authors [37-39], arbitrarily used the term ‘hyperfibrinolysis’ for lysis greater than a certain maximal amplitude on TEG/ROTEM testing (Schöchl uses EXTEM ML/1h > 15%). However, confusion has arisen with this viscoelastometric-associated terminology because traditionally hyperfibrinolysis describes a situation in which fibrinolytic activity is greater than fibrin formation, clot integrity is threatened, and there is clot breakdown [40], rather than a loose term used simply to describe increased evidence of fibrinolysis [38,41-44]. Therefore, the term ‘TEG/ROTEM hyperfibrinolysis’ has been suggested in relation to the viscoelastometric measurements [40]. In an attempt to combine the traditional hyperfibrinolysis definition with the viscoelastometric-associated terminology, and for the purpose of this study, we decided to define hyperfibrinolytic a ROTEM trace only when the EXTEM LI60 exceeded the reference interval and the fibrinogen concentration was ≤ 100 mg/dL. If the fibrinogen concentration was > 100 mg/dL, it was defined as “increased fibrinolysis”. The rationality was to align the definition of viscoelastometric hyperfibrinolysis, based on increased evidence of fibrinolysis at 60 min [33,41,43,44], with heighted risk of bleeding, as suggested by the traditional hyperfibrinolysis definition. A fibrinogen concentration ≤ 100 mg/dL was chosen as an index of "increased risk of bleeding", as fibrinogen concentrations below this value were associated with a high annual percentage of spontaneous bleeding in human patients with afibrinogenemia or hypofibrinogenemia [45]. The fibrinogen cutoff concentration was based on human data due to the lack of similar studies in dogs and the comparable physiological fibrinogen concentrations in these 2 species [46]. Using our ROTEM definition of increased fibrinolysis and hyperfibrinolysis, we demonstrated that dogs presenting with intracavitary effusions not only had a smaller EXTEM A5 and LI60 with higher ML/2h, but also a more severe grade of fibrinolysis compared to the controls. Again, this result did not change when animals with clinically relevant bleeding were excluded from analysis from both groups. Because these 2 groups of dogs were matched for type of underlying disease, it is possible, that the higher grade of fibrinolysis observed in these dogs was due to the presence of intracavitary fluid, as previously suggested in both humans and dogs [11-15]. However, our study was not designed to demonstrate that the presence of an intracavitary effusion was the cause of this result. In fact, the presence of the intracavitary effusion could be just a marker identifying dogs with a more severe disease, with the severity being the cause of the enhanced fibrinolysis. This could be a possibility because, in our matching strategy for type of underlying disease, we have not matched the dogs also by disease severity due to the lack of a scoring system applicable to all the included illnesses. However, in a previous study we demonstrated that, at least in dogs with congestive heart failure, it was not the severity of the cardiac disease but the presence of ascites that was associated with, and therefore the possible cause, of hyperfibrinolysis [11].

One advantage of studying fibrinolysis by ROTEM assay compared to FDPs and D-dimer concentrations is that it allows to recognize 3 patterns of fibrinolysis [33]. Our results showed that in dogs with intracavitary effusion late, intermediate and fulminant fibrinolysis were present in 18.8%, 12.5% and 3.1% of the cases, respectively. By contrast, only 9.4% of the control dogs had a late fibrinolysis, while none of them had intermediate or fulminant fibrinolysis. These results clearly showed that dogs with intracavitary effusion had an increased frequency and a more severe ROTEM pattern of fibrinolysis compared to dogs without intracavitary effusions. Again, this result was confirmed also when animals with clinically relevant bleeding were excluded from analysis from both groups. Although this study, and our previous studies [11-13], found that clinically relevant bleeding was more frequent in dogs with intracavitary effusion, most hemorrhage resulted from rupture of pathological/neoplastic organs or secondary to trauma. The lack of spontaneous bleeding, despite enhanced fibrinolysis, may be explained by the low frequency of fulminant fibrinolysis, which is the ROTEM pattern of fibrinolysis associated with the worse outcome in humans [33,47]. Moreover, when fulminant fibrinolysis is present, bleeding may not occur in the absence of trauma or rupture of a pathological organs [48], even if the hyperfibrinolysis lead to severe hypofibrinogenemia [49]. Due to the retrospective nature of this study, it was not possible to establish if the increased/hyper fibrinolysis preceded or followed the bleeding event; nevertheless, from a biological point of view it was likely that hypotension and accumulation of intracavitary fluid, which both followed the bleeding episode, triggered the fibrinolysis [11-15,29,33,34,50]. It is certainly possible that the increased/hyper fibrinolysis could have then contributed to the magnitude of the bleeding events, threatening clots formation and stability. Beyond the increased/hyper fibrinolysis as a possible cause for the higher frequency of clinically relevant bleeding in dogs with intracavitary effusion, the design of our study might also have played a part in this result. In fact, by default, all dogs with intracavitary hemorrhage were included in the studied group and not in the controls.

A second advantage of studying fibrinolysis by ROTEM assay compared to obtaining FDPs and D-dimer concentrations is that it allows dogs with hypofibrinolysis to be distinguished from those with basal fibrinolysis. Although an investigation of hypofibrinolysis is beyond the scope of this study, it should be noted that, at least in humans, hypofibrinolysis seems to play a major role in the pathophysiology of myocardial infarction, thrombosis, sepsis, and disseminated intravascular coagulation [28], and that the presence of a hypofibrinolytic state in some clinical conditions has been associated with poorer outcomes [37-39]. Therefore, its identification may be important despite viscoelastometric tests have been considered unsuitable for the detection of hypofibrinolysis by some authors [51].

The secondary aim of this study was to describe the grade of severity of fibrinolysis detected by ROTEM and by FDPs, D-dimer and fibrinogen concentrations, and to assess the degree of agreement between these 2 classification schemes. In a manner similar to how we aligned the traditional hyperfibrinolysis definition with viscoelastometric-associated terminology, we also graded fibrinolysis as increased fibrinolysis or hyperfibrinolysis based on a combination of the semiquantitative FDPs, D-dimer and fibrinogen concentrations. A fibrinogen concentration equal/below or above 100 mg/dL, respectively, was set as the cut-off values for the 2 grades of fibrinolysis. Similarly to the ROTEM findings, also the use of FDPs and D-dimer showed that dogs with intracavitary effusions have a lesser degree of hypofibrinolysis and basal fibrinolysis, and a higher degree of hyperfibrinolysis compared to controls, and that this result was still present excluding dogs with clinically relevant bleeding from statistical analysis. Nevertheless, the overall agreement between the 2 classification schemes was poor, with most disagreement being caused by dogs classified as hypofibrinolytic or with basal fibrinolysis by ROTEM as having an increased fibrinolysis when classified by FDPs and D-dimer concentrations (Table 6). A possible explanation for this discrepancy is that while ROTEM assay can detect severe fibrinolysis, it is not sensitive enough to detect less severe forms of fibrinolysis [52-54]. Alternatively, plasma semiquantitative FDPs concentrations might have potentially overestimated the real grade of fibrinolysis present in our dogs. In fact, dogs with increased fibrinolysis were so classified due to their increased concentrations in FDPs (Table 5). Because this was the only semiquantitative and manually performed test in our study, it is possible that the use of a more robust quantitative and automated FDPs assay could have led to different results. A final hypothesis for the discrepancy between ROTEM assays and FDPs and D-dimer concentrations is that while ROTEM detects increased/hyper fibrinolysis as long as the production of plasmin within the general circulation overwhelms the neutralizing capacity of the antiplasmin, the FDPs and D-dimer concentrations suggest the presence of an increased/hyper fibrinolysis after its onset and beyond its duration, due to the time needed for the production of these products and their subsequent clearance to basal concentrations [48]. Euglobulin lysis time is a test of global fibrinolysis, and it has been used for estimating the functional fibrinolytic capacity of plasma and as gold standard in assessing fibrinolysis [41,55,56]. However, in the absence of this test, or any other third test for evaluating fibrinolysis (e.g. plasmin-α2-antiplasmin complex), it was not possible to determine whether the ROTEM assay or the FDPs and D-dimer concentrations misclassified the real grade of fibrinolysis severity in our dogs.

One advantage of studying fibrinolysis by FDPs, D-dimer and fibrinogen concentrations compared to ROTEM assay is that it allows to distinguish primary versus secondary increased/hyper fibrinolysis. This distinction, which cannot be extrapolated from ROTEM assay results, is crucial for decisions on antifibrinolytic treatment in patients with clinically relevant hyperfibrinolysis [4]. At the moment rapid evaluation of the current status of the fibrinolytic system remains a challenge, making decision on antifibrinolytic treatment, for example in human trauma patients where degree and type of fibrinolysis are very variable, still empiric [57]. In our study, while there was a poor (i.e., 34.4%) overall agreement between ROTEM and plasma semiquantitative FDPs, D-dimer and fibrinogen concentrations in assessing grade of severity of fibrinolysis, this percentage rose to 40% when we only considered cases of possible hyperfibrinolysis (Table 6). Therefore, it might be suggested to bypass the empiric use of tranexamic acid in patient with fibrinolysis, performing in parallel both ROTEM and plasma determination of semiquantitative FDPs, D-dimer and fibrinogen. If both classification methods indicated the presence of hyperfibrinolysis, then semiquantitative FDP and D-dimer concentrations could be used in guiding antifibrinolytic therapy only for the cases of primary hyperfibrinolysis.

There are other 2 limitations to our study. First, in attempting to study the relevance of intracavitary effusion on the fibrinolytic system, we matched dogs in the study group (group 1) with those in a control population (group 2), based on type of disease. In doing so, we end up with a statistically significant increased number of sexually intact males with intracavitary effusions compared to the controls. It was possible that the variations in testosterone concentrations between the 2 groups could have influenced the results. Decreases in testosterone concentrations in humans are associated with an enhancement of fibrinolytic inhibition via increased synthesis of the plasminogen activator inhibitor-1 [58]. However, in all our previous studies we found enhanced fibrinolytic activity in dogs with intracavitary effusions, despite the control populations being 100% matched for gender and sexual status [11-13]. Second, none of the dogs in our study population had variables in extrinsically activated assays without aprotinin (EXTEM) compared with corresponding variables from extrinsically activated thromboelastometric assays with aprotinin (APTEM, TEM Innovations GmbH, Munich, Germany), as suggested for early diagnosis of fibrinolysis [41,59-61]. Nevertheless, a recent study in humans found that fibrinolysis seen in EXTEM tracings was a reliable finding and that APTEM assays failed to improve the accuracy of the diagnosis [29].

Conclusions

In summary, ROTEM assay results supported our previous findings suggesting the presence of an enhanced fibrinolytic state in dogs with intracavitary effusions [11-13]. Moreover, dogs with intracavitary effusions showed an increased frequency, and a different and more severe pattern of fibrinolysis, compared to dogs without intracavitary effusions. An enhanced fibrinolytic state in dogs with intracavitary effusion was also detected by determining plasma semiquantitative FDPs, D-dimer and fibrinogen concentrations. The overall agreement between the 2 classification schemes was poor. Further studies need to be carried out to determine if concurrent use of the 2 classification methods in selected cases might aid decision making on antifibrinolytic agent administration in patient with hyperfibrinolysis.

Minimal anonymized data set.

Excel dataset of the patients’ clinical records included in the present study.

(XLSX)

Click here for additional data file.

25 Jul 2019

PONE-D-19-15389

Enhanced fibrinolysis detection in a natural occurring canine model with intracavitary effusions: comparison and degree of agreement between thromboelastometry and FDPs, D-dimer and fibrinogen concentrations

PLOS ONE

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Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The authors should include healthy values for comparison obtained in the same conditions. Further, the presence of bleeding in both groups of studied animals makes difficult to interpret the results of the ROTEM analysis.

The presentation of the results in the figures should be optimized. The graphs could be combined in one figure with different panels. Note that there is a typo in figure one ("intracavitay").

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2. We noticed you have some minor occurrence of overlapping text with the following previous publications, which needs to be addressed:

-'Hemostatic findings of pleural fluid in dogs and the association between pleural effusions and primary hyperfibrino(geno)lysis: A cohort study of 99 dogs', https://doi.org/10.1371/journal.pone.0192371

-'Evaluation of rotation thrombelastography for the diagnosis of hyperfibrinolysis in trauma patients', https://doi.org/10.1093/bja/aen083

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

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We note that one or more of the authors are employed by a commercial company: San Marco Veterinary Clinic, Padua, Italy.

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Reviewer #1: This is a well written manuscript with well described methods and statistics. My major concern about this study is the heterogenous patient population, which I am concerned limits the value of the data analysis.

The basic premise of the study is that patients with peritoneal and pleural effusions are more likely to have increased fibrinolysis than patients without effusions, but the authors present no rationale for this hypothesis. While they cite their previous work documenting traditional coagulation and fibrinolysis assays that support the concept, I am perplexed by the mechanisms that would underly this finding, and the authors do not attempt to explain why a dog with right sided heart failure would develop hyperfibrinolysis. Given that 10/23 dogs with effusions had hemoperitoneum, I am concerned that the findings in this study are driven largely by the dogs that bled. A statistical analysis (or at least descriptive statistics) showing how many of the dogs that demonstrated hyperfibrinolysis were hemoperitoneum cases and how many were not bleeding should be done to determine if all of these findings are driven by the bleeding dogs. I would argue that combining all of these types of disease processes into a single group makes these findings very difficult to interpret. Also having no data on the severity of the clinical signs and degree of shock in the bleeding patients makes it difficult to decide how to use the results of this study since pervious work has shown that the severity of shock is closely associated with viscoelastic measures of hyperfibrinolysis in dogs (Fletcher et al., JVECC, 2016). I worry that the small number of cases in this study and the heterogeneity of the group make the findings difficult to interpret and impossible to generalize. Without some sort of statistical analysis demonstrating that the hemoperitoneum cases are not solely driving these findings, I cannot endorse the conclusions of the authors.

The classification scheme proposed to categorize fibrinolysis in these patients is interesting and potentially useful, but I have some concerns that with the very small number of dogs in this study, these numerous classifications are confusing and of questionable benefit in the study. The arbitrary cutoffs for the ROTEM and traditional parameters evaluated would need to be validated in a larger study, and I worry that this complex scheme detracts from the findings of the paper. I would suggest that using a more simplified scheme denoting patients as hyperfibrinolytic, with normal systemic fibrinolysis, and hypofibrinolytic would improve the clarity of this paper.

Reviewer #2: In this veterinary clinical study, the authors select two groups of animals from a large cohort considering the presence of cavitary effusion. Their aim is to study the fibrinolytic status of these animals in relation to the presence of pleural, peritoneal, or pericardial effusions by ROTEM technology. The study has several limitations that should be considered by the authors. They do not have a healthy control study group to compare their results on sick animals. The authors label their control group as "Sick dogs without intracavitary effusion". However, in both groups the animals are sick. It would be important to have an adequate healthy group for comparison. The presence of bleeding in a portion of the individuals in both groups complicates the interpretation. The authors should include an analysis of their results if these animals are excluded.

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29 Aug 2019

RE: Manuscript PONE-D-19-15389

Enhanced fibrinolysis detection in a natural occurring canine model with intracavitary effusions: comparison and degree of agreement between thromboelastometry and FDPs, D-dimer and fibrinogen concentrations

Dear Dr. Pablo Garcia de Frutos,

The authors would like to thank You and the Reviewers for the time spent and for their constructive comments in attempt to improve our manuscript.

A number of changes (listed below) have been made to the current manuscript to incorporate the suggestions you made and to address the comments of the Reviewers. We hope that these are satisfactory to allow for publication. Belows follow our reply to your 3 specific requests.

1) The authors should include healthy values for comparison obtained in the same conditions.

Authors’ reply: To give an idea for comparison to healthy values, reference intervals (obtained from healthy animals) for all the parameters studied were included (and still are) in the result section of our paper. On the other hand, if the Editor meant that we should include a healthy control population, as requested from Reviewer #2, we cannot adhere to this request because it would heavily damage the validity of our study (please see below).

Case–control is a type of epidemiological observational study where subjects are observed in order to determine both their exposure and outcome status. A common misconception among investigators is that case-control design is perceived as a comparison between a group of diseased individuals versus a group of “normal” (i.e., disease-free) individuals. This conceptualization of the case-control design is inconsistent with the actual goal of control group selection, which is to provide a representative sample of the risk factors distribution among the population at risk. Therefore, following the current epidemiology textbooks indications and international guidelines on observational studies, we have included as a control population a group of sick dogs as much as possible similar to the specific diseased group (cases), but without the specific outcome of interest that we wanted to study (i.e., the presence of intracavitary effusion). On the other hand, a control group of healthy animals would have weakened the study, because different for too many aspects from a diseased group and not even representative of the general population. Selection of healthy subjects has been heavily criticized in the modern literature and the so-called “healthy patient bias” has been coined (REFERENCES follow).

1) Bloom MS, Schisterman EF, Hediger ML. Selecting controls is not selecting “normals”: design and analysis issues for studying the etiology of polycystic ovary syndrome. Fertil Steril. 2006;86(1):2-12

2) Lewallen S, Courtright P. Epidemiology in practice: case-control studies. Comm Eye Health 1998;11:57-58

3) Wacholder JK, Silverman JS, McLaughlin JK, Mandel JS. Selection of controls in case-control studies. II. Types of Controls. Am J Epidemiol; 1992, 135:1029-1041

4) Shrank WH, Patrick AR, Brookhart A. Healthy user and related biases in observational studies of preventive interventions: a primer for physicians. J Gen Intern Med, 2011;26(5):546-550

2) Further, the presence of bleeding in both groups of studied animals makes difficult to interpret the results of the ROTEM analysis.

Authors’ reply: To address your concern additional statistical analysis excluding bleedings dogs from both groups were performed as requested. The additional analyses showed that bleeding is not the solely driving of our finding. In fact, excluding these dogs none of the results changed. Please see the 'Revised Manuscript with Track Changes’ at:

• Materials and methods lines: 214-220

• Results: lines 269-271; 279-283; 357-360; 364-367; 372-374; Table 1, 3, 4, and 5.

• Discussion: 427–430; 462–462; 484–485; 520–521.

3) The presentation of the results in the figures should be optimized. The graphs could be combined in one figure with different panels. Note that there is a typo in figure one ("intracavitay").

Authors’ reply: Following your instruction the 5 figures have been combined in one figure with 5 panels. The typo mistake has been corrected.

Kind regards,

Andrea Zoia, DVM, Cert SAM, Dip ECVIM-CA

(Corresponding Author)

PS: If you have any further questions or comments, please do not hesitate to contact me.

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Authors’ reply: following the above PDFs instructions, numerous changes have been made to the article’s format. If we still do not meet Plos One’s style requirements we would be grateful to receive more specific suggestions.

2. We noticed you have some minor occurrence of overlapping text with the following previous publications, which needs to be addressed:

-'Hemostatic findings of pleural fluid in dogs and the association between pleural effusions and primary hyperfibrino(geno)lysis: A cohort study of 99 dogs', https://doi.org/10.1371/journal.pone.0192371

-'Evaluation of rotation thrombelastography for the diagnosis of hyperfibrinolysis in trauma patients', https://doi.org/10.1093/bja/aen083

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

Authors’ reply: Overlapping test in figure caption of this paper with our previous work “'Hemostatic findings of pleural fluid in dogs and the association between pleural effusions and primary hyperfibrino(geno)lysis: A cohort study of 99 dogs” was present and it has been drastically reduced/eliminated. There does remain some overlap between this article and the above article only for the author affiliation and for the references, which from our understanding should not constitute an issue.

Small overlapping test in the introduction of this paper with the 'Evaluation of rotation thrombelastography for the diagnosis of hyperfibrinolysis in trauma patients' had been rephrased as requested.

3. Thank you for stating the following in the Competing Interests/Financial Disclosure* (delete as necessary) section:

"The author(s) received no specific funding for this work"

We note that one or more of the authors are employed by a commercial company: San Marco Veterinary Clinic, Padua, Italy.

a) Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

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Authors’ reply: The San Marco Veterinary Clinic (Padua, Italy) did not play any role in the study nor altered our adherence to all PLOS ONE policies on sharing data and materials. An updated Funding Statement and a Competing Interests Statement has been included in the cover letter, as requested.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Authors’ reply: none requested

Reviewer #2: Partly

Authors’ reply: please see our reply to your comments below.

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Authors’ reply: additional statistical analysis excluding dogs with bleeding has been added as requested (see our reply to your comments below).

Reviewer #2: N/A

Authors’ reply: none requested

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Authors’ reply: none requested

Reviewer #2: Yes

Authors’ reply: none requested

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Authors’ reply: none requested

Reviewer #2: Yes

Authors’ reply: none requested

________________________________________

5. Review Comments to the Author

Reviewer #1:

1) This is a well written manuscript with well described methods and statistics. My major concern about this study is the heterogenous patient population, which I am concerned limits the value of the data analysis.

Authors’ reply: We thank the Reviewer to find our manuscript well written and with appropriate description on methods and statistics. To address your concern on the heterogenous patient population further statistical analysis excluding bleedings dogs from both groups were performed. For details see our reply to your point number 3.

2) The basic premise of the study is that patients with peritoneal and pleural effusions are more likely to have increased fibrinolysis than patients without effusions, but the authors present no rationale for this hypothesis. While they cite their previous works documenting traditional coagulation and fibrinolysis assays that support the concept, I am perplexed by the mechanisms that would underlay this finding, and the authors do not attempt to explain why a dog with right sided heart failure would develop hyperfibrinolysis.

Authors’ reply: We thank the Reviewer for this comment which allowed us to improve our article. In our previous works cited in the current article there is well explained mechanism that would justify why dogs with peritoneal and pleural effusions are more likely to have increased fibrinolysis than patients without effusions, including dogs with right sided heart failure. It is true that this information is not present in the current article. Therefore, we briefly summarized rationale for this hypothesis also in this article following your comment, leaving the references to acquire more in-depth details for the readers more interested in such pathophysiological information. Moreover further 12 references (references 35 to 46) have been added to support this hypothesis. Please see 'Revised Manuscript with Track Changes’ at lines 465-468.

3) Given that 10/23 dogs with effusions had hemoperitoneum, I am concerned that the findings in this study are driven largely by the dogs that bled. A statistical analysis (or at least descriptive statistics) showing how many of the dogs that demonstrated hyperfibrinolysis were hemoperitoneum cases and how many were not bleeding should be done to determine if all of these findings are driven by the bleeding dogs. I would argue that combining all of these types of disease processes into a single group makes these findings very difficult to interpret. Also having no data on the severity of the clinical signs and degree of shock in the bleeding patients makes it difficult to decide how to use the results of this study since pervious work has shown that the severity of shock is closely associated with viscoelastic measures of hyperfibrinolysis in dogs (Fletcher et al., JVECC, 2016). I worry that the small number of cases in this study and the heterogeneity of the group make the findings difficult to interpret and impossible to generalize. Without some sort of statistical analysis demonstrating that the hemoperitoneum cases are not solely driving these findings, I cannot endorse the conclusions of the authors.

Authors’ reply: We thank the Reviewer for this comment that allow us to improve our article. To address your concern additional statistical analysis excluding bleedings dogs from both groups were performed as requested. The additional analyses showed that bleeding is not the solely driving of our finding. In fact, excluding these dogs none of the results changed. Please see 'Revised Manuscript with Track Changes’ at:

• Materials and methods lines: 214-220

• Results: lines 269-271; 279-283; 357-360; 364-367; 372-374; Table 1, 3, 4, and 5.

• Discussion: 427–430; 462–462; 484–485; 520–521.

4) The classification scheme proposed to categorize fibrinolysis in these patients is interesting and potentially useful, but I have some concerns that with the very small number of dogs in this study, these numerous classifications are confusing and of questionable benefit in the study. The arbitrary cutoffs for the ROTEM and traditional parameters evaluated would need to be validated in a larger study, and I worry that this complex scheme detracts from the findings of the paper. I would suggest that using a more simplified scheme denoting patients as hyperfibrinolytic, with normal systemic fibrinolysis, and hypofibrinolytic would improve the clarity of this paper.

Authors’ reply: We thank the Reviewer to find the classification scheme proposed to categorize fibrinolysis in our patients interesting and potentially useful. Nevertheless, we realize that you have some concern with the very small number of dogs in this study being analysed in multiple subgroups. However, while in the descriptive statistics we have several groups (hypofibrinolysis, basal fibrinolysis, increased fibrinolysis, and hyerfibrinolys, with the latter two groups sometimes dived in primary and secondary) in our inferential statistics we used, to simplify the classification system, only 3 groups of fibrinolysis. In fact, hypo and basal fibrinolysis are always grouped together and for the same reasons we also kept together dogs with primary and secondary increased fibrinolysis and primary and secondary hyperfibrinolysis. These information are now better spelled/explained in the captions of tables 3, 4, 5, and 6.

Our cut-offs for the ROTEM and traditional parameters are not as “arbitrary” as you state. In fact, in part they are justify throughout the paper with biological explanations (i.e., fibrinogen) and in part they are in line with human and veterinary studies where fibrinolysis cut-offs are based on reference intervals. Nevertheless, we do agree with you that, as any new classification system, replication studies need to be performed to validate our proposed classification system.

Finally, we cannot adhere to your suggestion of using a more simplified scheme including only the hyperfibrinolytic, the normal fibrinolytic and the hypofibrinolytic state for two reasons:

a) It would change partially the target of our study. In fact, we also aim to try to differentiate patient with increased fibrinolysis that are at risk of bleeding (hyperfibrinolysis) from patient with increased fibrinolysis that are probably not at risk of bleeding (increased fibrinolysis). In addition, we also try to differentiate primary from secondary fibrinolysis. Unfortunately, we would not be able to do these types of differentiations with the scheme from you proposed.

b) As a LI60 of 100% essentially encompasses the normal range, some authors consider the ROTEM in essence unsuitable to define hypofibrinolysis (although this is done extensively in human’s trauma research). For this reason, we decided in our inferential statistics to combine the hypo and the basal fibrinolysis in a single group, leaving this distinction only for the descriptive statistics.

REFERENCE: Lisman T. Decreased Fibrinolytic Capacity in Cirrhosis and Liver Transplantation Outcomes. Liver Transpl. 2019 Mar;25(3):359-361.

Reviewer #2:

1) In this veterinary clinical study, the authors select two groups of animals from a large cohort considering the presence of cavitary effusion. Their aim is to study the fibrinolytic status of these animals in relation to the presence of pleural, peritoneal, or pericardial effusions by ROTEM technology. The study has several limitations that should be considered by the authors. They do not have a healthy control study group to compare their results on sick animals. The authors label their control group as "Sick dogs without intracavitary effusion". However, in both groups the animals are sick. It would be important to have an adequate healthy group for comparison.

Authors’ reply: To give an idea for comparison to a healthy population, reference intervals (obtained from healthy animals) for all the parameters studied were included (and still are) in the result section of our paper. On the other hand, a true healthy control group cannot be added, as requested from You, because it would heavily damage the validity of our study (please see below).

Case–control is a type of epidemiological observational study where subjects are observed in order to determine both their exposure and outcome status. A common misconception among investigators is that case-control design is perceived as a comparison between a group of diseased individuals versus a group of “normal” (i.e., disease-free) individuals. This conceptualization of the case-control design is inconsistent with the actual goal of control group selection, which is to provide a representative sample of the risk factors distribution among the population at risk. Therefore, following the current epidemiology textbooks indications and international guidelines on observational studies, we have included as a control population a group of sick dogs as much as possible similar to the specific diseased group (cases), but without the specific outcome of interest that we wanted to study (i.e., the presence of intracavitary effusion). On the other hand, a control group of healthy animals would have weakened the study, because different for too many aspects from a diseased group and not even representative of the general population. Selection of healthy subjects has been heavily criticized in the modern literature and the so-called “healthy patient bias” has been coined (REFERENCES follow).

1) Bloom MS, Schisterman EF, Hediger ML. Selecting controls is not selecting “normals”: design and analysis issues for studying the etiology of polycystic ovary syndrome. Fertil Steril. 2006;86(1):2-12

2) Lewallen S, Courtright P. Epidemiology in practice: case-control studies. Comm Eye Health 1998;11:57-58

3) Wacholder JK, Silverman JS, McLaughlin JK, Mandel JS. Selection of controls in case-control studies. II. Types of Controls. Am J Epidemiol; 1992, 135:1029-1041

4) Shrank WH, Patrick AR, Brookhart A. Healthy user and related biases in observational studies of preventive interventions: a primer for physicians. J Gen Intern Med, 2011;26(5):546-550

2) The presence of bleeding in a portion of the individuals in both groups complicates the interpretation. The authors should include an analysis of their results if these animals are excluded.

Authors’ reply: We thank the Reviewer for this comment that allow us to improve our article. To address your concern additional statistical analysis excluding bleedings dogs from both groups were performed as requested. The additional analyses showed that bleeding is not the solely driving of our finding. In fact, excluding these dogs none of the results changed. Please see 'Revised Manuscript with Track Changes’ at:

• Materials and methods lines: 214-220

• Results: lines 269-271; 279-283; 357-360; 364-367; 372-374; Table 1, 3, 4, and 5.

• Discussion: 427–430; 462–462; 484–485; 520–521.

________________________________________

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Authors’ reply: none requested

Reviewer #2: No

Authors’ reply: none requested

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

30 Sep 2019

PONE-D-19-15389R1

Enhanced fibrinolysis detection in a natural occurring canine model with intracavitary effusions: comparison and degree of agreement between thromboelastometry and FDPs, D-dimer and fibrinogen concentrations

PLOS ONE

Dear Mr Zoia,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The authors have addressed the concerns adequatedly. For clarity, it would be interesting that some changes in the organization of the section are considered, as suggested by the reviewers.

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We look forward to receiving your revised manuscript.

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Pablo Garcia de Frutos

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Thank you for mostly addressing the major concerns raised in my previous review. I have a few additional minor comments that I would recommend the authors address before publication.

(1) Thank you for better describing the rationale behind the study regarding the potential contribution of effusions to systemic hyperfibrinolysis. Given that this is the main point of the study, I would recommend that you move that description from the Discussion to the Introduction.

(2) Your manuscript as it stands does not include any hypotheses. In the interest of informing the reader about the rationale for your statistical analyses, it would be appropriate to rewrite the final paragraph of your introduction to reframe the objectives of your study as specific, testable hypotheses that are linked back to your statistical analyses.

Best of luck with your manuscript.

Reviewer #2: The authors have answered the criticisms on the initial version of the manuscript. The laboratory reference intervals could be included in the relevant tables and figure.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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23 Oct 2019

RE: Manuscript PONE-D-19-15389R1

Enhanced fibrinolysis detection in a natural occurring canine model with intracavitary effusions: comparison and degree of agreement between thromboelastometry and FDPs, D-dimer and fibrinogen concentrations

Dear Dr. Pablo Garcia de Frutos,

The authors would like to thank You and the Reviewers for the time spent and for their constructive comments in attempt to improve our manuscript.

Few changes (listed below) have been made to the current manuscript to incorporate the suggestions of the Reviewers in the organization of the section of the manuscript. We hope that these are satisfactory to allow for publication.

Kind regards,

Andrea Zoia, DVM, Cert SAM, Dip ECVIM-CA

(Corresponding Author)

PS: If you have any further questions or comments, please do not hesitate to contact me.

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Authors’ reply: none requested

Reviewer #2: All comments have been addressed

Authors’ reply: none requested

________________________________________

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Authors’ reply: none requested

Reviewer #2: Yes

Authors’ reply: none requested

________________________________________

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Authors’ reply: none requested

Reviewer #2: Yes

Authors’ reply: none requested

________________________________________

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Authors’ reply: none requested

Reviewer #2: Yes

Authors’ reply: none requested

________________________________________

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Authors’ reply: none requested

Reviewer #2: Yes

Authors’ reply: none requested

________________________________________

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Thank you for mostly addressing the major concerns raised in my previous review. I have a few additional minor comments that I would recommend the authors address before publication.

(1) Thank you for better describing the rationale behind the study regarding the potential contribution of effusions to systemic hyperfibrinolysis. Given that this is the main point of the study, I would recommend that you move that description from the Discussion to the Introduction.

Authors’ reply: The relevant paragraphs has been moved from the discussion ('Revised Manuscript with Track Changes’ at lines: 442 - 446) to the introduction as requested ('Revised Manuscript with Track Changes’ at lines: 63 - 68)

.

(2) Your manuscript as it stands does not include any hypotheses. In the interest of informing the reader about the rationale for your statistical analyses, it would be appropriate to rewrite the final paragraph of your introduction to reframe the objectives of your study as specific, testable hypotheses that are linked back to your statistical analyses.

Best of luck with your manuscript.

Authors’ reply: The final paragraph has been re-written stating our 3 hypotheses of the study. i.e.:

1) ROTEM could detect an enhanced and more severe pattern of fibrinolysis in dogs with intracavitary effusions compared to dogs without effusion.

2) There was an agreement in fibrinolysis severity detection between the combination of ROTEM assay results and fibrinogen concentrations, or alternatively by concentrations of FDPs, D-dimers and fibrinogen.

3) Dogs with intracavitary effusions had more primary fibrinolysis/hyperfibrinolysis compared to dogs without effusion when assessed by concentrations of FDPs, D-dimers and fibrinogen.

Please see the 'Revised Manuscript with Track Changes’ at lines: 82 – 95.

Reviewer #2: The authors have answered the criticisms on the initial version of the manuscript. The laboratory reference intervals could be included in the relevant tables and figure.

Authors’ reply: The laboratory reference intervals have been included in the relevant tables and figure as requested. Please see 'Revised Manuscript with Track Changes’ at: table 1 and 2 + Fig 1.

________________________________________

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Authors’ reply: none requested

Reviewer #2: No

Authors’ reply: none requested

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

30 Oct 2019

Enhanced fibrinolysis detection in a natural occurring canine model with intracavitary effusions: comparison and degree of agreement between thromboelastometry and FDPs, D-dimer and fibrinogen concentrations

PONE-D-19-15389R2

Dear Dr. Zoia,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Pablo Garcia de Frutos

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

8 Nov 2019

PONE-D-19-15389R2

Enhanced fibrinolysis detection in a natural occurring canine model with intracavitary effusions: comparison and degree of agreement between thromboelastometry and FDPs, D-dimer and fibrinogen concentrations

Dear Dr. Zoia:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE.

With kind regards,

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on behalf of

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Academic Editor

PLOS ONE